Dilution-to-Extinction Culturing Combined with Catalase Supplementation Yielded Diverse Previously-uncultured Oligotrophic Bacteria from Lake Soyang

초록

High-Throughput-Culturing method (HTC) based on dilution-to-extinction has been improved in various ways for the efficient isolation of unstably growing or uncultured bacterial groups. In a recent study, catalase supplementation into a culture medium as a hydrogen peroxide scavenger showed a great enhancement of cellular growth of previously uncultivated freshwater bacterioplankton, the acI clade. In this study, the catalase-amended HTC was applied to surface lake water collected from Lake Soyang every three months from November 2016 to August 2017. Cells in the water samples were diluted with sterilized lake water with a final density of 2 cells/ml and aliquoted into each well in multiwell plates with 10 U/ml of catalase. The multiwell plates were incubated at 20°C for 4 weeks and microbial growth was measured using a flow cytometer. Phylogenetic analysis of 16S rRNA gene showed that catalase-amended HTC yielded many oligotrophic bacterial isolates belonging to diverse freshwater bacterioplankton clade, such as acI, acIV, and LD28. Particularly, bacterial strains belonging to the acI and acIV clades accounted for approximately 25% of growth-positive strains. This result showed that acI and acIV clades of Actinobacteria, known to be abundant in freshwater environment but difficult to be isolated, can be isolated more efficiently by the simple catalase-amended dilution-to-extinction culturing method. [Supported by a grant of the Mid-Career Research Program through the NRF]